Fig. 6

Amphotericin B reduced the expression of CD163, CD204, CD206 and B7-H1, and enhanced the phagocytic function of co-cultured macrophages. a Raji cells were co-cultured with macrophages treated with DMSO or amphotericin B for 72 h. iNOS, CD163, CD204 and CD206 protein expression in macrophages were determined by flow cytometry. Amphotericin B reduced the expression of M2-related phenotypes CD163, CD204 and CD206 on the surface of the co-cultured macrophages (P < 0.05). b Cells were treated as a, and the protein levels of iNOS, CD163, CD204 and CD206 in macrophages were determined by immunoblotting. c Cells were treated as a, and the mRNA expression of IL10, IL-12 and TNF-α in the co-cultured macrophages was detected by RT-PCR (P < 0.01). d Amphotericin B enhanced the phagocytic function of macrophages. e Amphotericin B reduced B7-H1 expression on the surface of co-cultured macrophages (P < 0.05). f Amphotericin B reduced B7-H1 protein expression in co-cultured macrophages. g Confocal microscopy revealed that amphotericin B reduced B7-H1 expression in co-cultured macrophages (600×). Scale 50 μm. h Raji cells were co-cultured with macrophages treated with DMSO or amphotericin B for 72 h. Then, these macrophages were co-cultured with CD3+ T cells in 24-well plates with or without 10 mg/ml of anti-B7-H1 (clone MIH1) or mouse IgG1 isotype control mAbs (eBioscience). After 72 h, CD3+ T cells were harvested for cell proliferation assay using a BrdU Flow Kit. Co-cultured immature macrophages significantly inhibited CD3+ T cell proliferation. Macrophages treated with amphotericin B restored CD3+ T cell proliferation (P < 0.05), which is consistent with the results of the anti-B7-H1 treatment. i Cells were treated as h, and CD4+ T cells were harvested for the detection of the percentage of CD4 + CD25 + Foxp3+ T cells by flow cytometry. Co-cultured macrophages significantly induced Foxp3 expression in Treg cells. Amphotericin B treatment and anti-B7-H1 antibody inhibited the expression of Foxp3 in Treg cells co-cultured with macrophages (P < 0.05), which was consistent with the results of the anti-B7-H1 treatment. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001